The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak P AN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtb PW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.How to cite this paper: Nascimento, D.V.,